Microfluidic device adapted for post-centrifugation use with selective sample extraction and methods for its use

ABSTRACT

The present disclosure relates to microfluidic devices adapted for post-centrifugation use with selective sample extraction, and methods for their use. Certain embodiments make use of a dye-selective sample extraction. Other embodiments make use of a geographically-selective sample extraction. Other embodiments are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication No. 61/223,412, which was filed Jul. 7, 2009. The presentapplication also claims the benefit of U.S. Provisional Application No.61/223,413, filed Jul. 7, 2009. Both of these applications areincorporated herein by reference in their entireties

TECHNICAL FIELD OF THE INVENTION

The present disclosure relates generally to microfluidic cytometrysystems and, more particularly, to a microfluidic device adapted forpost-centrifugation use with selective sample extraction and methods forits use.

BACKGROUND OF THE INVENTION

Flow cytometry-based cell sorting was first introduced to the researchcommunity more than 20 years ago. It is a technology that has beenwidely applied in many areas of life science research, serving as acritical tool for those working in fields such as genetics, immunology,molecular biology and environmental science. Unlike bulk cell separationtechniques such as immuno-panning or magnetic column separation, flowcytometry-based cell sorting instruments measure, classify and then sortindividual cells or particles serially at rates of several thousandcells per second or higher. This rapid “one-by-one” processing of singlecells has made flow cytometry a unique and valuable tool for extractinghighly pure sub-populations of cells from otherwise heterogeneous cellsuspensions.

Cells targeted for sorting are usually labeled in some manner with afluorescent material. The fluorescent probes bound to a cell emitfluorescent light as the cell passes through a tightly focused, highintensity, light beam (typically a laser beam). A computer recordsemission intensities for each cell. These data are then used to classifyeach cell for specific sorting operations. Flow cytometry-based cellsorting has been successfully applied to hundreds of cell types, cellconstituents and microorganisms, as well as many types of inorganicparticles of comparable size.

Flow cytometers are also applied widely for rapidly analyzingheterogeneous cell suspensions to identify constituent sub-populations.Examples of the many applications where flow cytometry cell sorting isfinding use include isolation of rare populations of immune system cellsfor AIDS research, isolation of genetically atypical cells for cancerresearch, isolation of specific chromosomes for genetic studies, andisolation of various species of microorganisms for environmentalstudies. For example, fluorescently labeled monoclonal antibodies areoften used as “markers” to identify immune cells such as T lymphocytesand B lymphocytes, clinical laboratories routinely use this technologyto count the number of “CD4 positive” T cells in HIV infected patients,and they also use this technology to identify cells associated with avariety of leukemia and lymphoma cancers.

Recently, two areas of interest are moving cell sorting towardsclinical, patient care applications, rather than strictly researchapplications. First is the move away from chemical pharmaceuticaldevelopment to the development of biopharmaceuticals. For example, themajority of novel cancer therapies are now biologics containing proteinsor peptides. These include a class of antibody-based cancertherapeutics. Cytometry-based cell sorters can play a vital role in theidentification, development, purification and, ultimately, production ofthese products.

There is also a move toward the use of cell replacement therapy forpatient care. Much of the current interest in stem cells revolves arounda new area of medicine often referred to as regenerative therapy orregenerative medicine. These therapies may often require that largenumbers of relatively rare cells be isolated from sample patient tissue.For example, adult stem cells may be isolated from bone marrow oradipose tissue and ultimately used as part of a re-infusion back intothe patient from whom they were removed. Cytometry lends itself verywell to such therapies.

There are two basic types of cell sorters in wide use today. They arethe “droplet cell sorter” and the “fluid switching cell sorter.” Thedroplet cell sorter utilizes micro-droplets as containers to transportselected cells to a collection vessel. The micro-droplets are formed bycoupling ultrasonic energy to a jetting stream. Droplets containingcells selected for sorting are then electrostatically steered to thedesired location. This is a very efficient process, allowing as many as90,000 cells per second to be sorted from a single stream, limitedprimarily by the frequency of droplet generation and the time requiredfor illumination.

A detailed description of a prior art flow cytometry system is given inUnited States Published Patent Application No. US 2005/0112541 A1 toDurack et al.

Droplet cell sorters, however, are not particularly biosafe. Aerosolsgenerated as part of the droplet formation process can carrybiohazardous materials. Because of this, biosafe droplet cell sortershave been developed that are contained within a biosafety cabinet sothat they may operate within an essentially closed environment.Unfortunately, this type of system does not lend itself to the sterilityand operator protection required for routine sorting of patient samplesin a clinical environment.

The second type of flow cytometry-based cell sorter is the fluidswitching cell sorter. Most fluid switching cell sorters utilize apiezoelectric device to drive a mechanical system which diverts asegment of the flowing sample stream into a collection vessel. Comparedto droplet cell sorters, fluid switching cell sorters have a lowermaximum cell sorting rate due to the cycle time of the mechanical systemused to divert the sample stream. This cycle time, the time betweeninitial sample diversion and when stable non-sorted flow is restored, istypically significantly greater than the period of a droplet generatoron a droplet cell sorter. This longer cycle time limits fluid switchingcell sorters to processing rates of several hundred cells per second.For the same reason, the stream segment switched by a fluid cell sorteris usually at least ten times the volume of a single micro-drop from adroplet generator. This results in a correspondingly lower concentrationof cells in the fluid switching sorter's collection vessel as comparedto a droplet sorter's collection vessel.

Newer generation microfluidics technologies offer great promise forimproving the efficiency of fluid switching devices and providing cellsorting capability on a chip similar in concept to an electronicintegrated circuit. Many microfluidic systems have been demonstratedthat can successfully sort cells from heterogeneous cell populations.They have the advantages of being completely self-contained, easy tosterilize, and can be manufactured on sufficient scales (with theresulting manufacturing efficiencies) to be considered a disposablepart.

A generic microfluidic device is illustrated in FIG. 1 and indicatedgenerally at 10. The microfluidic device 10 comprises a substrate 12having a fluid flow channel 14 formed therein by any convenient processas is known in the art. The substrate 12 may be formed from glass,plastic or any other convenient material, and may be substantiallytransparent or substantially transparent in a portion thereof. Incertain embodiments, the substrate 12 is injection molded. In certainembodiments, the substrate 12 comprises industrial plastic such as aCyclo Olefin Polymer (COP) material, or other plastic. As a result, thesubstrate 12 is transparent such that a cytometry optics module cananalyze the sample fluid stream as described further below. In oneembodiment, the microfluidic device 10 is disposable.

The substrate 12 further has three ports 16, 18 and 20 coupled thereto.Port 16 is an inlet port for a sheath fluid. Port 16 has a central axialpassage that is in fluid communication with a fluid flow channel 22 thatjoins fluid flow channel 14 such that sheath fluid entering port 16 froman external supply (not shown) will enter fluid flow channel 22 and thenflow into fluid flow channel 14. The sheath fluid supply may be attachedto the port 16 by any convenient coupling mechanism as is known to thoseskilled in the art. In one embodiment, the sheath fluid comprises abuffer or buffered solution. For example, the sheath fluid comprises0.96% Dulbecco's phosphate buffered saline (w/v), 0.1% BSA (w/v), inwater at a pH of about 7.0.

Port 18 also has a central axial passage that is in fluid communicationwith a fluid flow channel 14 through a sample injection tube 24. Sampleinjection tube 24 is positioned to be coaxial with the longitudinal axisof the fluid flow channel 14. Injection of a liquid sample of cells intoport 18 while sheath fluid is being injected into port 16 will thereforeresult in the cells flowing through fluid flow channel 14 surrounded bythe sheath fluid. The dimensions and configuration of the fluid flowchannels 14 and 22, as well as the sample injection tube 24 are chosenso that the sheath/sample fluid will exhibit laminar flow as it travelsthrough the device 10, as is known in the art. Port 20 is coupled to theterminal end of the fluid flow channel 14 so that the sheath/samplefluid may be removed from the microfluidic device 10.

While the sheath/sample fluid is flowing through the fluid flow channel14, it may be analyzed using cytometry techniques by shining anillumination source through the substrate 12 and into the fluid flowchannel 14 at some point between the sample injection tube 24 and theoutlet port 20. Additionally, the microfluidic device 10 could bemodified to provide for a cell sorting operation, as is known in theart.

Although basic microfluidic devices similar to that describedhereinabove have been demonstrated to work well, there is a need in theprior art for improvements to cytometry systems employing microfluidicdevices. The present invention is directed to meeting this need.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to microfluidic devices adapted forpost-centrifugation use with selective sample extraction, and methodsfor their use. Certain embodiments make use of a dye-selective sampleextraction. Other embodiments make use of a geographically-selectivesample extraction.

In certain embodiments, a method of analyzing a sample fluid in amicrofluidic cytometry system is disclosed, the method comprising thesteps of a) supplying a sample to a sample well in a microfluidicdevice; b) centrifuging the microfluidic device to create a first samplelayer and a second sample layer within said sample well; c) extractingfirst fluid from said first sample layer; and d) analyzing saidextracted first fluid using cytometry while said extracted first fluidis in the microfluidic device.

In other embodiments, a microfluidic device is disclosed, comprising asample well having a sample input port for receiving a sample into themicrofluidic device, said sample well having a first end and a secondend, and a sample output port in fluid communication with said samplewell, said sample output port located between said first end and saidsecond end, wherein sample may be withdrawn through said sample outputport to form a withdrawn sample and said withdrawn sample is notwithdrawn at said first end or at said second end.

In further embodiments, a microfluidic device is disclosed, comprising asample well having a sample input port for receiving a sample into themicrofluidic device, said sample well having a first end and a secondend, a first sample output port in fluid communication with said samplewell, said first sample output port located between said first end andsaid second end, and a second sample output port in fluid communicationwith said sample well, said second sample output port located betweensaid first sample output port and said second end, wherein sample may bewithdrawn through said first sample output port to form a firstwithdrawn sample and said first withdrawn sample is not withdrawn atsaid first end or at said second end, and wherein sample may bewithdrawn through said second sample output port to form a secondwithdrawn sample and said second withdrawn sample is not withdrawn atsaid first end or at said second end.

Other embodiments are also disclosed.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a prior art microfluidic device.

FIG. 2 is a schematic front view of a microfluidic device according to afirst embodiment of the present disclosure.

FIG. 3 is a schematic front view of a microfluidic device according to asecond embodiment of the present disclosure.

FIG. 4 is a schematic front view of a microfluidic device according to athird embodiment of the present disclosure.

FIG. 5 is a schematic front view of a microfluidic device according to afourth embodiment of the present disclosure.

DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

For the purposes of promoting an understanding of the principles of thedisclosure, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill nevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alterations and furthermodifications in the illustrated device, and such further applicationsof the principles of the disclosure as illustrated therein arecontemplated as would normally occur to one skilled in the art to whichthe disclosure relates.

The present disclosure is generally directed to systems for theseparation and/or analysis of a biological sample on a microfluidicdevice using cytometry (such as flow cytometry or image cytometry). Toincrease the efficiency of cytometry operations, it is desirable tostart with a sample containing mostly those types of cells that aredesired to be individually studied or isolated. One method known in theart is to subject the sample to centrifugation prior to cytometryanalysis. After centrifugation, the sample components will be separatedinto layers. Due to factors such as mass, density and specific gravity,the desired component, or cell population, can then be more easilyextracted. This process is common laboratory practice and many commonsample containers and centrifuge devices are commercially available forthis purpose. However, this procedure introduces the possibility thatthe sample layers will become remixed during the transfer from theinitial collection vessel to the microfluidic device. By adapting themicrofluidic device itself to be suitable for containing the sampleduring the centrifugation process, the possibility of layer mixing aftercentrifugation and prior to analysis by the cytometer is reduced. Thisalso eliminates the need for multiple containers and reduces thepossibility of outside contaminates being introduced into the sample (orpotentially harmful sample components being released into the outsideenvironment). Since the microfluidic device can be manufactured to besterile (e.g. exposed to gamma irradiation), the entire process ofcentrifugation, cytometry measurement, and sorted fraction collectioncan be conducted in a closed, sterile environment. Most importantly, thecentrifugation process and transfer of material among containers canintroduce loss of 10% or more of the cells. When low cell numbers areinvolved this loss is intolerable, in fact it is impossible to applyeven microcentrifugation in many cases where sample volume is low.

Dye-Selective Sample Extraction

In one embodiment, FIG. 2 illustrates a device 200 which is configuredto achieve the separation of biological components contained within asample. The device 200 includes a sample well 202 for receiving a sample204 from an external source (not shown) via port 206. In certainembodiments, the sample well 202 is elongated to facilitate gradientseparation of the sample components when the device 200 is placed in acentrifuge. Various chemical additives known in the art and commonlyused to facilitate the process of separation based on the density ofsample constituents, such as Ficoll-Hypaque to name just onenon-limiting example, may be added to the sample 204 to increase theseparation effect. In certain embodiments, such chemical additives maybe added to the sample well 202 during manufacturing, eliminating theneed for the user to add them during the sampling process. In addition,in certain embodiments a dye may be added to the Ficoll-Hypaque toprovide a color distinction between the layers as described hereinbelow.

As will be appreciated by those skilled in the art, the microfluidicdevice may be subjected to the forces of centrifugation in eitherdirection along the longitudinal axis of the sample well 202. With theFicoll-Hypaque method, the cells are layered on to the Ficoll-Hypaquesolution, so that the cells are “on top” relative to the force ofcentrifugation. In other embodiments and other additives, the entiresituation might be inverted. By using the microfluidic device as thecontainer for the sample before analysis, during centrifugation, duringanalysis and/or after sorting, the user may move the small sample from acentrifuge in one part of the laboratory to a cytometer somewhere else,and then after analysis the sample may be transported to a thirdlocation, all in a sterile environment.

Channel 208 is connected via port 210 at one end of sample well 202 toretrieve the desired sample component after centrifugation. As onenon-limiting example, human blood may be injected into the sample well202 after which the device 200 is subjected to centrifugation toseparate the mononuclear cells 212, i.e. lymphocytes and monocytes, fromthe larger granulocytic cells 214 and red blood cells 216. Aftercentrifugation, the port 210 may be opened to allow the sample 204 toflow through channel 208 to a cytometry analysis section 218 withindevice 200 for further cytometric analysis and/or separation. Forexample, the cytometry analysis section 218 may image the cells in theflow channel prior to switching element 223 and then divert a subset ofthe mononuclear cells previously isolated, by identifying them usingimage or common fluorescence-based methods, and then exercisingappropriate control of a switching element 223, into an extraction well222 and undesirable cells into a waste well 224. In certain embodiments,the wells 222 and 224 communicate with ports (not shown) to allowextraction of their contents from the microfluidic device 200. Thespecific analysis and/or sorting performed in analysis section 218 isnot critical to the present disclosure and may be performed in a varietyof different manners in different embodiments.

In certain embodiments, the sorting performed by the cytometry analysissection 218 is greater than a binary sort. In other embodiments, thesorting performed by the cytometry analysis section 218 is greater thana tertiary sort. In other embodiments, the sorting performed by thecytometry analysis section 218 is greater than a quartnery sort. Inexecuting the sort, certain embodiments comprise at least five channels.Other embodiments comprise at least ten channels. Other embodimentscomprise at least 100 channels. Other embodiments comprise at least 256channels.

The flow through channel 208 may be initiated by capillary action orother microfluidic flow means known in the art. Such methods may requirethat additional channels or features (not shown) be included in themicrofluidic device. In one embodiment, sample cells are added to thesample well 202 first and then the Ficoll-Hypaque solution is added. Themicrofluidic device is then subjected to centrifugation with the appliedforce being toward the port 206. As the sample flows through the channel208, the lighter mononuclear cells 212 will be drawn off first. Thecytometry analysis and sorting section 218 is programmed to close theport 210 or, in alternative embodiments, a valve 220, upon the detectionby analysis section 218 of the particular dye contained within theFicoll-Hypaque solution. In this case the dye will be in the same layeras the plasma-Ficoll interface which is just below the layer oflymphocytes. The granulocytic cells 214 and red blood cells 216 would beheld below the plasma-Ficall interface layer and, therefore, the dye.This allows the full amount of mononuclear cells 212 contained in thesample 204 to be extracted while still rejecting the undesiredcomponents within the gradient separation. In a second example, necroticcells can easily be separated without any chemical additives due totheir higher density. In addition to blood, the present disclosurecontemplates that other types of biological or chemical samples may beanalyzed in this manner, it being understood that the present disclosureis useful with any sample that can be separated by centrifugation.

When dye is added to the sample solution, it must be done in such a waythat the dye ends up in the correct layer after centrifugation. Incertain embodiments, the dye may be designed to have the correct densityto achieve this. In other embodiments, the dye can be bonded at themolecular level with the Ficall-Hypaque (or other chemical additive) ina manner that does not negatively impact the specific gravity of theFicoll-Hypaque.

In other embodiments, various density gradient chemicals, each withtheir own specific gravity and/or dye color, can be used to detect andextract individual components from a sample. For example, as shown inFIG. 3, the cytometry analysis section 318 may be configured to divertsorted cells or particles (such as viral particles) from a first samplecomponent into a first extraction well 322 until the detection of afirst colored dye corresponding to the density gradient chemical layer(which holds a second sample component 304). The device 300 is similarto the device 200, and like reference designators are used to designatelike components. After centrifugation, the cytometry analysis section318 will adjust the analysis parameters to account for the new type ofcells being analyzed and divert sorted cells into a second extractionwell 326. Upon the detection of a second colored dye solution (whichholds a third sample component 306), the cytometry analysis section 318will then adjust the analysis parameters to account for the new type ofcells being analyzed and divert all remaining cells into the waste well324 and/or close valve 220 to cease sample flow. In certain embodiments,the wells 322, 324 and 326 communicate with ports (not shown) to allowextraction of their contents from the microfluidic device 300. Thespecific analysis and/or sorting performed in analysis section 318 isnot critical to the present disclosure and may be performed in a varietyof different manners in different embodiments.

In certain embodiments, the sorting performed by the cytometry analysissection 318 is greater than a binary sort. In other embodiments, thesorting performed by the cytometry analysis section 318 is greater thana tertiary sort. In other embodiments, the sorting performed by thecytometry analysis section 318 is greater than a quartnery sort. Inexecuting the sort, certain embodiments comprise at least five channels.Other embodiments comprise at least ten channels. Other embodimentscomprise at least 100 channels. Other embodiments comprise at least 256channels.

In certain embodiments, the microfluidic devices comprise two or morepieces. The two pieces are coupled together using any desirable meanssuch as, by way of non-limiting example, a thermal bonding process, anultrasonic welding process or an adhesive process. In one embodiment thetwo pieces are halves. In another embodiment, the two pieces are dividedasymmetrically in the plane, e.g. a planar cover and a piece containingchannels. In still other embodiments, multiple pieces are assembled.Other ways of coupling the two pieces will be readily apparent to oneskilled in the art depending upon the material used to fabricate themicrofluidic device.

Geographically-Selective Sample Extraction

FIG. 4 illustrates another embodiment, in which a device 400 which isconfigured to achieve the separation of biological components containedwithin a sample. The device 400 is similar to the device 200, and likereference designators are used to designate like components. The device400 includes a sample well 202 for receiving a sample 204 from anexternal source (not shown) via port 206. The sample well 202 iselongated to facilitate gradient separation of the sample componentswhen the device 400 is placed in a centrifuge. Various chemicaladditives known in the art, such as Ficoll-Hypaque, may be added to thesample 204 to increase the separation effect as described hereinabove.In certain embodiments, such chemical additives may be added to thesample well 202 during manufacturing, eliminating the need for the userto add them during the sampling process.

Channel 208 is connected via port 210 at a specific location along thelength of sample well 202 to retrieve the desired sample component aftercentrifugation. The exact location of the port 210 is determined by thenature of the sample being processed and the component desired to beextracted, such that the placement of the port 210 is designed to be inclose physical proximity to the position of the component desired to beextracted after centrifugation. This ensures that a maximum volume ofthe desired sample component may be extracted from the sample well 202.It will be appreciated, therefore, that the placement of the port 210 inthe device 400 is made strategically to more precisely align with thelocation of the desired sample component than the device 200 illustratedin FIG. 2.

As one non-limiting example, human blood may be injected into the samplewell 202 after which the device 400 is subjected to centrifugation toseparate the mononuclear cells 212, i.e. lymphocytes and monocytes, fromthe larger granulocytic cells 214 and red blood cells 216. Aftercentrifugation, the port 210 may be opened to allow the sample 204 toflow through channel 208 to a cytometry analysis section 218 withindevice 400 for further cytometric analysis and/or separation. Forexample, the cytometry analysis section 218 may divert sorted desirablecells, by appropriate control of the switching device 223, into anextraction well 222 and undesirable cells into a waste well 224. Incertain embodiments, undesirable cells may also be expelled from thedevice 400 through a waste port (not shown). The specific analysisand/or sorting performed in analysis section 218 is not critical to thepresent disclosure.

The flow through channel 208 may be initiated by capillary action orother microfluidic pumping means known in the art. As the sample flowsthrough the channel 208, the lighter mononuclear cells 212 will be drawnoff first due to their proximity to the port 210. In certainembodiments, multiple channels 208 and multiple ports 210 may beconnected along the sample well 202 within the region of the desiredsample component (such as mononuclear cells 212) to facilitate morecomplete extraction of the desired sample component. The cytometryanalysis section 218 is programmed to close the port 210 or, inalternative embodiments, a valve 220, after a certain amount of samplefluid or cells has been extracted. This amount depends on the volume ofthe sample 204, the nature of the sample being processed, and theexpected volume of the desired component layer.

For simplicity and ease of illustration, the presently illustratedembodiments show single channels extending between the components, areasor sections of the illustrated devices. However, it should beappreciated that the single channels may be representative of multiplecytometry channels and a variety of possible configurations of channelsas would occur to one skilled in the art.

In other embodiments, multiple channels can be used to extract multiplecomponents from a sample. The addition of two or more types ofFicoll-Hypaque (or other appropriate additive) can also be used tocreate additional layers in the centrifuged sample. For example, asshown in FIG. 5, a device 500 is provided into which an analysis sample202 may be loaded through port 206. By the use of appropriate additivesas discussed herein, the analysis sample may, for example, be dividedinto three layers 302, 304 and 306 post-centrifugation. The device 500is adapted to selectively extract sample from the layers 302 and 304 bypositioning ports 310 a and 310 b, respectively, in alignment with thelayers 302 and 304. Port 310 a and or valve 320 a may be opened to allowsample 302 to flow through the channel 308 a. A cytometry analysissection 318 may be configured to divert sorted cells from the firstsample component 302 into a first extraction well 322 by control ofvalve 323. Similarly, undesired cells from the first sample component302 may be diverted to waste well 324 by appropriate control of thevalve 323. Once the cytometry section 318 determines that analysis ofthe first sample component 302 is complete, the cytometry analysissection 318 is programmed to close the port 310 a or, in alternativeembodiments, a valve 320 a.

The cytometry analysis section 318 will then adjust the analysisparameters to account for the new type of cells being analyzed, openport 310 b, allowing fluid from the second sample component 304 to flowto the cytometry analysis section 318, and divert sorted cells into asecond extraction well 326 by appropriate control of valve 323. Once thecytometry section 318 determines that analysis of the second samplecomponent 304 is complete, the cytometry analysis section 318 isprogrammed to close the port 310 b or, in alternative embodiments, avalve 320 b.

It will be appreciated that by proper location of the ports 310 a and310 b, the device 500 may take advantage of the multiple layers ofsample post-centrifugation. Analysis and sorting of the samplecomponents is made easier by separating the sample components intolayers using centrifugation, and then extracting sample components in apurer form by means of strategic location of the extraction ports 310.It will be appreciated by those skilled in the art that any number ofextraction ports may be utilized to more precisely extractpost-centrifugation samples from the analysis sample.

While the disclosure has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiments have been shown and described and thatall changes and modifications that come within the spirit of thedisclosure are desired to be protected.

1. A method of analyzing a sample fluid in a microfluidic cytometrysystem, the method comprising the steps of: a) supplying a sample to asample well in a microfluidic device; b) centrifuging the microfluidicdevice to create a first sample layer and a second sample layer withinsaid sample well; c) extracting first fluid from said first samplelayer; and d) analyzing said extracted first fluid using cytometry whilesaid extracted first fluid is in the microfluidic device.
 2. The methodof claim 1, further comprising the steps of: e) sorting a first portionof said extracted first fluid into a first well based upon resultsobtained in step (d); and f) sorting a second portion of said extractedfirst fluid into a second well based upon results obtained in step (d).3. The method of claim 2, wherein said sorting at steps (e) and (f) isselected from the group consisting of: greater than a binary sort,greater than a tertiary sort, greater than a quaternary sort.
 4. Themethod of claim 2, wherein said sorting at steps (e) and (f) employs apredetermined number of microfluidic channels, said predetermined numberselected from the group consisting of: at least five channels, at leastten channels, at least 100 channels, and at least 256 channels.
 5. Themethod of claim 2, further comprising the steps of: g) extracting secondfluid from said second sample layer; and h) analyzing said extractedsecond fluid using cytometry while said extracted second fluid is in themicrofluidic device.
 6. The method of claim 5, further comprising thestep of: i) sorting a third portion of said extracted second fluid intoa third well based upon results obtained in step (h).
 7. The method ofclaim 5, wherein: said extracted first fluid is extracted from saidsample well at a first location; and said extracted second fluid isextracted from said sample well at a second location, said secondlocation being different than said first location.
 8. The method ofclaim 1, further comprising the step of: e) adding a chemical additiveto said sample well prior to performing step (a).
 9. The method of claim1, further comprising the step of: e) adding Ficoll-Hypaque to saidsample well prior to performing step (a).
 10. The method of claim 1,further comprising the step of: e) adding a dye to said sample wellprior to performing step (a).
 11. The method of claim 1, wherein step(a) further comprises supplying a sample of human blood to a sample wellon a microfluidic device
 12. A microfluidic device, comprising: a samplewell having a sample input port for receiving a sample into themicrofluidic device, said sample well having a first end and a secondend; and a sample output port in fluid communication with said samplewell, said sample output port located between said first end and saidsecond end; wherein sample may be withdrawn through said sample outputport to form a withdrawn sample and said withdrawn sample is notwithdrawn at said first end or at said second end.
 13. The microfluidicdevice of claim 12, further comprising: a sorting well; a waste well;and a channel coupled to said sorting well and said waste well; a valvedisposed in said channel for directing fluid flowing in said channelinto either said sorting well or said waste well.
 14. The microfluidicdevice of claim 12, further comprising a chemical additive disposed inthe sample well prior to receiving a sample.
 15. The microfluidic deviceof claim 14, wherein said chemical additive comprises Ficoll-Hypaque.16. The microfluidic device of claim 14, wherein said chemical additivecomprises a dye.
 17. A microfluidic device, comprising: a sample wellhaving a sample input port for receiving a sample into the microfluidicdevice, said sample well having a first end and a second end; a firstsample output port in fluid communication with said sample well, saidfirst sample output port located between said first end and said secondend; and a second sample output port in fluid communication with saidsample well, said second sample output port located between said firstsample output port and said second end; wherein sample may be withdrawnthrough said first sample output port to form a first withdrawn sampleand said first withdrawn sample is not withdrawn at said first end or atsaid second end; and wherein sample may be withdrawn through said secondsample output port to form a second withdrawn sample and said secondwithdrawn sample is not withdrawn at said first end or at said secondend.
 18. The microfluidic device of claim 17, further comprising: afirst sorting well; a second sorting well; a waste well; and a channelcoupled said first sorting well, said second sorting well and said wastewell; a valve disposed in said channel for directing fluid flowing insaid channel into either said first sorting well, said second sortingwell or said waste well.
 19. The microfluidic device of claim 17,further comprising a chemical additive disposed in the sample well priorto receiving a sample.
 20. The microfluidic device of claim 19, whereinsaid chemical additive comprises Ficoll-Hypaque.
 21. The microfluidicdevice of claim 19, wherein said chemical additive comprises a dye.